The acidification of clathrin-coated vesicles from bovine brain has been shown to be mediated by a proton translocating ATPase which operates in parallel with a chloride transporter. A similar system has been identified in vesicles prepared from bovine renal medulla. The purification and reconstitution of the proton pump and chloride transporter from renal medulla is the central aim of this proposal; attempts at achieving this will be modeled after similar experiments performed with the clathrin-coated vesicle acidification system. Such a degree of resolution will allow for definition of any possible subunits and will provide a pure system with which to examine the stoichiometry and potential reversibility of the proton pump. With reconstitution of the chloride transporter, patch clamping of the proteoliposome will be performed to determine if the chloride transporter is a channel conductance. Moreover, with coreconstitution of the proton pump and chloride transporter, the precise interrelationship of these proteins will be determined. Characterization of the effects of mineralocorticoids on the proton pump and chloride transporter from rabbit renal medulla vesicles will be expanded. Any potential modulators of the rabbit vesicular acidification system will be tested with the reconstituted bovine kidney proton pump and chloride transporter to define function in a purified system.